Project summary description Influenza virus infections affect millions of people worldwide every year and cause serious mortality. Current treatment options are limited to viral strain-specific vaccination and are problematic due to antiviral drug resistance. There is an urgent need to identify novel host innate immune mechanisms providing broad range protection against influenza. Bronchial epithelial cells orchestrate an oxidative extracellular antimicrobial system present in the airway surface liquid consisting of the protein lactoperoxidase (LPO), the thiocyanate anion (SCN- ) and hydrogen peroxide (H2O2). LPO oxidizes SCN- using H2O2 into hypothiocyanite (OSCN-) that has known in vitro antiviral effects. Dual oxidase 1 (Duox1), an NADPH oxidase highly expressed in bronchial epithelial cells, is the H2O2 source for the system. Our long-term goal is to determine whether the Duox1/H2O2/LPO/SCN- antiviral system could be manipulated in influenza infection for therapeutic purposes in human patients. The objective of this proposal is to determine and characterize the antiviral role of Duox1 and LPO against influenza in multiple experimental systems. Our preliminary data show that 1) primary bronchial epithelial cells inactivate several influenza viruses in an Duox1/H2O2/LPO/SCN- -dependent manner, 2) Duox1-deficient mice have increased mortality and morbidity, impaired viral clearance and leukocyte recruitment following influenza infection in vivo, and 3) the in vitro influenza-inactivating effect of this mechanism can be enhanced to inhibit influenza infection. Based on these data, our central hypothesis is that the Duox1/H2O2/LPO/SCN- system attenuates influenza infection, both in vitro and in vivo, and can be boosted to fight influenza. The rationale for the proposed research is that there is a need to better understand how powerful the antiviral Duox1/LPO-based system is and how can it be manipulated for therapeutic purposes. The main hypothesis will be tested in cell- free, airway epithelial and mouse model systems using a wide range of influenza strains. It is anticipated that our aims will yield several impactful outcomes including 1) detailed description of the anti-influenza mechanism of action of the Duox1/H2O2/LPO/SCN- system; 2) determination of the in vivo relevance of Duox1 in fighting a wide range of influenza strains; and 3) exploring the therapeutic potential of the Duox1/H2O2/LPO/SCN- system to improve influenza clearance and to diminish associated lung damage. Our innovative work shows that the Duox1/H2O2/LPO/SCN- system inactivates influenza, and uses a Duox1-deficient mouse strain for in vivo studies. The significance of the outlined work relies in establishing the relevance of a novel innate immune mechanism of the airways that can be enhanced to attenuate influenza infections or applied in conjunction with influenza vaccines to potentially enhance efficacy. In summary, our proposed work will have a positive impact in the fields of airway epithelial biology and antiviral innate immune responses by identifying Duox1 and LPO, as novel, crucial weapons of the bronchial epithelium against influenza.